The proposals in this application fall into three areas: I. We shall investigate the stereochemical course of reactions of phosphate monoesters in order to provide an unabmiguous stereochemical basis for the interpretation of the mechanisms of phosphokinases and other phosphoryl transfer enzymes. To discriminate amongst the three formally equivalent oxygens of a phosphate monoester (thus creating the chirality at phosphorus) we are adopting two strategies: one uses sulphur, 16 O and 18 O, and the other employs 16 O, 17 O, and 18 O. II. In order to relate enzyme structure to enzyme function and to watch the "evolutionary" development of catalytic function, we are isolated mutant E. coli beta-lactamases that derive from selection for a particular functional change in the enzyme. We intend to correlate the sequence changes (the consequential structural changes being interpreted using the crystal structure) with the measured alterations in catalytic behavior. III. As part of a program to elucidate the mechanism of enzyme-catalyzed beta-lactam hydrolysis (this being an increasingly serious problem in the treatment of bacterial infection) we propose to study the mechanistic pathways that result in beta-lactamase inactivation by the recently discovered compound, calvulanic acid. This is currently the only available beta-lactamase inactivator, and elucidation of its action (which appears to be of the 'suicide' type) is a matter of considerable urgency and importance in the antibacterial field.